Skip to main content
SpringerLink
Log in

Purification of Holstein Bull Semen Paraoxonase 1 (PON1) by Hydrophobic Interaction Chromatography and Investigation of Its Inhibition Kinetics by Heavy Metals

  • Published:
Biological Trace Element Research Aims and scope Submit manuscript

Abstract

In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu2+, Mn2+, Cd2+, Zn2+, Ni2+, and Pb2+, on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10−5 M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd2+, inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu2+, Mn2+, Zn2+, Ni2+, and Pb2+ were found as 2.59 × 10−3, 1.17 × 10−3, 42.74 × 10−3, 99.10 × 10−3, 48.80 × 10−3 mM, respectively. Conversely, Cd2+ increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu2+, Mn2+, Zn2+, Ni2+, and Pb2+ are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Arslan M, Erzengin M, Demir D (2011) Comparison of serum paraoxonase 1 (PON1) activities among different sheep breeds in Turkey. J Anim Vet Adv 10(4):489–494

    Article  CAS  Google Scholar 

  2. Mackness MI, Walker CH (1988) Multiple forms of sheep serum A-esterase activity associated with the high-density lipoprotein. Biochem J 250:539–545

    CAS  PubMed Central  PubMed  Google Scholar 

  3. Mackness B, Durrington PN, Mackness MI (1998) Human serum paraoxonase. Gen Pharmacol 31:329–336

    Article  CAS  PubMed  Google Scholar 

  4. Kokouvaa M, Koureasa M, Dardiotisb E, Almpanidoub P, Kalogerakic A, Kyriakoud D, Hadjigeorgioub GM, Hadjichristodouloua C (2013) Relationship between the paraoxonase 1 (PON1) M55L and Q192R polymorphisms and lymphohaematopoietic cancers in a Greek agricultural population. Toxicology 307:12–16

    Article  Google Scholar 

  5. Khersonsky O, Tawfik D (2005) Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 44(16):6371–6382

    Article  CAS  PubMed  Google Scholar 

  6. Camps J, Marsillach J, Joven J (2009) The paraoxonases: role in human diseases and methodological difficulties in measurement. Crit Rev Clin Lab Sci 46(2):83–106

    Article  CAS  PubMed  Google Scholar 

  7. La Du BN, Aviram M, Billecke S, Navab M, Primo-Parmo S, Sorenson RC, Standiford TJ (1999) On the physiological role(s) of the paraoxonases. Chem Biol Interact 119–120:379–388

    PubMed  Google Scholar 

  8. Erzengin M, Basaran I, Cakir U, Aybey A, Sinan S (2012) In vitro inhibition effect of some dihydroxy coumarin compounds on purified human serum paraoxonase 1 (PON1). Appl Biochem Biotechnol 168:1540–1548

    Article  CAS  PubMed  Google Scholar 

  9. Aviram M, Rosenblat M (2004) Paraoxonases 1, 2, and 3, oxidative stress, and macrophage cell formation during atherosclerosis development. Free Radic Biol Med 37(9):1304–1316

    Article  CAS  PubMed  Google Scholar 

  10. Mackness MI (1989) Possible medical significance of human serum “A”-esterases. In: Reiner E, Aldridge WN, Hoskin FCG (eds) Enzymes hydrolysing organophosphorus compounds. Ellis Horwood, Chichester, pp 203–213

    Google Scholar 

  11. Mackness MI (1989) “A”-esterases: enzymes looking for a role? Biochem Pharmacol 38:385–390

    Article  CAS  PubMed  Google Scholar 

  12. Mackness MI, Durrington PN (1995) HDL, its enzymes and its potential to influence lipid peroxidation. Atherosclerosis 115:243–253

    Article  CAS  PubMed  Google Scholar 

  13. Stein O, Stein Y (1999) Atheroprotective mechanisms of HDL. Atherosclerosis 144:285–301

    Article  CAS  PubMed  Google Scholar 

  14. Hu H, McCally M (eds) (2002) Life support: the environment and human health (book review). J Sociol Soc Welf 4:1-9

    Google Scholar 

  15. Foote RH, Brockett CC, Kaproth MT (2002) Motility and fertility of bull sperm in whole milk extender containing antioxidants. Anim Reprod Sci 71:13–23

    Article  CAS  PubMed  Google Scholar 

  16. Stohs SJ, Baggihi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Med 18:321–336

    Article  CAS  Google Scholar 

  17. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–251

    Article  CAS  PubMed  Google Scholar 

  18. Gan KN, Smolen A, Eckerson HW, La Du BN (1991) Purification of human serum paraoxonase/arylesterase, evidence for one esterase catalyzing both activities. Drug Metab Dispos 19:100–106

    CAS  PubMed  Google Scholar 

  19. Sinan S, Kockar F, Arslan O (2006) Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglycoside derived antibiotics. Biochimie 88:565–574

    Article  CAS  PubMed  Google Scholar 

  20. Demir N, Nadaroğlu H, Demir Y (2008) Purification of human serum paraoxonase and effect of acetylsalicylic acid on paraoxonase activity in vitro and rat serum liver and heart in vivo. Pharm Biol 46(6):393–399

    Article  CAS  Google Scholar 

  21. Laemmli DK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–683

    Article  CAS  PubMed  Google Scholar 

  22. Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 57:685

    Google Scholar 

  23. Kara HE, Sinan S, Turan Y (2011) Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme. J Chromatogr B 879:1507–1512

    Article  CAS  Google Scholar 

  24. Samra ZQ, Shabir S, Rehmat Z, Zaman M, Nazir A, Dar N, Athar MA (2010) Synthesis of cholesterol-conjugated magnetic nanoparticles for purification of human paraoxonase 1. Appl Biochem Biotechnol 162:671–686

    Article  CAS  PubMed  Google Scholar 

  25. Pla A, Rodrigo L, Hernandez AF et al (2007) Effect of metal ions and calcium on purified PON1 and PON3 from rat liver. Chem Biol Interact 167:63–70

    Article  CAS  PubMed  Google Scholar 

  26. Erol K, Gençer N, Arslan M, Arslan O (2013) Purification, characterization, and investigation of in vitro inhibition by metals of paraoxonase from different sheep breeds. Artif Cells Blood Substit Biotechnol 41:125–130

    CAS  Google Scholar 

  27. Meeker JD, Rossano MG, Protas B, Diamond MP, Puscheck E, Daly D, Paneth N, Wirth JJ (2008) Cadmium, lead, and other metals in relation to semen quality: human evidence for molybdenum as a male reproductive. Tox Environ Health Perspect 116(11):1473–1479

    Article  CAS  Google Scholar 

  28. Gençer N, Arslan O (2009) Purification human PON1Q192 and PON1R192 isoenzymes by hydrophobic interaction chromatography and investigation of the inhibition by metals. J Chromatogr B 877:134–140

    Article  Google Scholar 

  29. Ekinci D, Beydemir Ş (2010) Purification of PON1 from human serum and assessment of enzyme kinetics against metal toxicity. Biol Trace Elem Res 135:112–120

    Article  CAS  PubMed  Google Scholar 

  30. Verit FF, Verit A, Ciftci H, Erel O, Celik H (2009) Paraoxonase-1 activity in subfertile men and relationship to other sperm parameters. J Androl 30:183–189

    Article  PubMed  Google Scholar 

  31. Massanyi P, Trandzik J, Nad P, Korenekova B, Skalicka M, Toman R, Lukac N, Halo M, Strapak P (2004) Concentration of copper, iron, zinc, cadmium, lead, and nickel in bull and ram semen and relation to the occurrence of pathological spermatozoa. J Environ Sci Health A 39(11–12):3005–3014

    Article  CAS  Google Scholar 

  32. Tvrda E, Knazıcka Z, Lukac N (2012) Selected heavy metals versus antioxidant parameters in bull seminal plasma—a comparative study. J Environ Sci Health A 47:1261–1266

    Article  CAS  Google Scholar 

  33. Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, Irvine DS (1998) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 59:1037–1046

    Article  CAS  PubMed  Google Scholar 

  34. Storey BT (1997) Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod 3:203–214

    Article  CAS  PubMed  Google Scholar 

Download references

Conflict of Interest

The authors report no conflicts of interest.

Author information

Authors and Affiliations

  1. Faculty of Science and Letters, Department of Chemistry, Balikesir University, 10100, Balikesir, Turkey

    Nurcan Dedeoğlu

  2. Susurluk Technical Vocational School of Higher Education, Balikesir University, 10100, Balikesir, Turkey

    Mikail Arslan

  3. Faculty of Science and Letters, Department of Chemistry, Aksaray University, 68100, Aksaray, Turkey

    Mahmut Erzengin

Authors
  1. Nurcan Dedeoğlu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mikail Arslan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mahmut Erzengin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mahmut Erzengin.

Additional information

The authors alone are responsible for the content and writing of the article.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dedeoğlu, N., Arslan, M. & Erzengin, M. Purification of Holstein Bull Semen Paraoxonase 1 (PON1) by Hydrophobic Interaction Chromatography and Investigation of Its Inhibition Kinetics by Heavy Metals. Biol Trace Elem Res 158, 29–35 (2014). https://doi.org/10.1007/s12011-014-9916-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12011-014-9916-8

Keywords

Search

Navigation